Method of controlling foliar microorganism populations

ABSTRACT

A method of controlling the population of a first microorganism at a foliar locus by preferentially enhancing the population of a second microorganism at said locus, comprises applying to the locus an amount of a durable selective habitat enhancer which substantially preferentially potentiates growth of said second microorganism with respect to said first microorganism. The durable selective habitat enhancer preferably comprises a substantially water-insoluble, weather resistant polymeric substrate and a binder which increases the durability of the habitat enhancer. The second microorganism can be endogenous to the foliar locus or exogenously applied. Chitin and cellulose are preferred habitat enhancers.

This application is a continuation of application Ser. No. 07/483,505,filed Feb. 23, 1990 which is now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a method of controlling the population of afirst microorganism at a foliar locus by preferentially enhancing thepopulation of a second microorganism at said locus.

Microbiological plant pathogens and insect pests of plants represent amajor source of loss of agricultural productivity throughout the world.Research on the biological control of foliar pathogens is limited whencompared with studies concerning this form of control for soilbornepathogens. However, as restrictions on the use of chemical pesticidesincrease, biological control of foliar pathogens is becoming moreimportant.

The ecosystems which exist on above-ground plant surfaces are complex,and the microbial interactions which occur on it are poorly understood.Plant pathogens and their interactions with the host plant have beeninfluenced under limited circumstances by the population of microbialepiphytes which exist in the foliar phylloplane. The introduction oforganisms that exhibit antagonism towards foliar pathogens in thisenvironment has been successful in controlling certain pathogens byseveral mechanisms, which include direct parasitism of the pathogen bythe epiphyte, competition for nutrients and space, the production ofantibiotics and/or lytic enzymes by the antagonist, and stimulation of ahost resistance response.

Recently, there have been several reported successes using introducedantagonists for the control of foliar diseases in the field. Spurr(1981) used Pseudomonas cepacia to inhibit the germination of Alternariaalternata and thereby control Alternaria leaf spot of tobacco in fieldtests by repeated applications over a period of three years. Limited butsignificant control of Cercospora leaf spot on peanut was achieved infield tests using aqueous suspensions of P. cepacia applied to foliageat two week intervals. In later experiments by Bailey and Spurr (1984),P. cepacia and Bacillus thuringiensis (Bt) were formulated as wettablepowders and sprayed onto peanut foliage using xanthan gum, awater-soluble polysaccharide, as a sticking agent. A decrease inCercospora leaf spot with both P. cepacia and Bt was observed. Knudsenand Spurr (1987) formulated several bacteria including B. cereus and P.cepacia into wettable powders and dusts and applied them to peanuts inthe field. Limited control of Cercospora arachidicola a was achievedusing P. cepacia although B. cereus survived in greater number, perhapsdue to its resistant spores.

An isolate of Bacillus subtilis was found to produce a compound thatinhibits urediniospore germination in Puccinia pelargonii-zonalis, thecausal agent of geranium rust (Rytter and Lukezic, 1986). A culturefiltrate applied to leaves prior to inoculation with the pathogenreduced the number of rust pustules by 75% compared to controls. Thedecrease in disease incidence resulting from the application of culturefiltrate indicated antibiotic activity. A fluorescent Pseudomonasisolated from the phylloplane of cacao (Theobroma cacao) exhibitedantagonism to Monilia roreri in vitro (Jimenez et al., 1986). Whentested in the field it achieved control similar to that obtained withfungicide treatments. Lindow (1983) has successfully used a geneticallyengineered ice-minus form of Pseudomonas syringae to compete withnaturally occurring ice nucleation-active bacteria and prevent frostdamage. The ice-minus mutant lacks the gene necessary to produce a largesurface protein that acts as an initiator for ice crystal formation.Mild frost damage, induced by nucleation-active bacteria, is a precursorfor many diseases. Thus, excluding the bacteria from themicroenvironment that they normally occupy reduces disease incidence.

The use of fungi as foliar biocontrol agents has received less attentionthan that of bacteria. There has, however, been success usingTrichoderma, which produces an array of potent antibiotics, and has beenshown to control Botyris bunch rot and Phomopsis viticola, the cause ofdead arm disease of grape (Gullino and Garibaldi, 1986). Fiveapplications of Trichoderma spores beginning at dormant bud stage werefound to be equivalent to the control obtained by a commercialfungicide.

It is to be noted that in each of these attempts at bacteriologicalbiocontrol of these various diseases, the introduced microorganismshowed poor long-term survivability. It is likely that the limitedsuccesses achieved utilizing these techniques were probably the resultof antibiotics present in the cultural filtrates coapplied to the plantswith the microorganisms.

The chemical environment encountered at the surface of a plant maygovern the degree of aggressiveness of virulence a pathogen exhibits.One focus of plant disease control using biological agents has been theintroduction of organisms capable of preempting the nutrient supply of apathogen (Baker and Cook, 1974). It has been shown that if anestablished epiphytic (foliar) ecosystem is continuously supplied with asource of nutrition, it becomes difficult for a newly introducedpathogen to compete and growth of the pathogen is limited accordingly(Warren, 1972).

Bashi and Fokkema (1977) showed that populations of Sporobolomycesroseus, an epiphytic yeast, increased when a 2% sucrose solution wasapplied to leaves as a food base. This indicated that the amount ofnutrients present on the leaf was a limiting factor with respect to thegrowth of these organisms. By monitoring the growth of yeasts on wheatleaves after the application of nutrients, Fokkema et al. (1975)demonstrated that epiphytic bacteria compete effectively for nutrients.They found that yeast populations increased only after antibiotics tolimit bacterial growth are applied with the nutrients. The ability ofbacteria to utilize certain nutrients varies. Danilewicz (1980) foundthat epiphytic bacteria capable of utilizing lignin precursors wereabundant where the precursors were available. The amount and type ofnutrients available on the leaf surface play an important role ininfluencing the growth of epiphytic bacteria. By studying the effect ofnutrients on the epiphytic bacteria of bean leaves, Morris and Rouse(1985) found that the application of low concentrations of nutrientsaffected the composition of the bacterial community by increasing theproportion of those bacteria able to utilize the nutrient applied.

In regard to disease control, addition of nutrients to aqueous bacterialsuspensions enhances survival of the bacterium. Control of northern leafblight of corn and early blight of tomato was thereby obtained ingreenhouse tests (Leben and Daft, 1965). Control of Phytophthorainfestans on tomato leaves in the greenhouse was achieved using waterextracts from composted organic wastes (Weltzien and Ketterer, 1986).Sterile-filtered or heat-treated extracts were ineffective. The use ofnitrogen-containing compounds such as urea and lecithin has also beeninvestigated. Burchill and Cook (1971) found that in apple leavesinfected with Venturia inaequalis, treatment with 5% urea resulted inthe fungus producing fewer ascospores. However, this was likely a resultof the urea promoting the rotting of the fallen leaves on the ground,thereby suppressing growth of the fungus. Ammonia, the breakdown productof urea, is toxic to some organisms and caused a rapid increase andsignificant shifts in the microbial populations from gram positivechromogens to gram negative nonchromogens. In vitro tests showed grampositive organisms stimulated growth of the scab fungus, while gramnegative organisms inhibited its growth. Boudrea and Andrews (1987) hadlittle or no success controlling V. inaequalis by the application ofChaetomium globosum ascospores to field-grown trees. The fact that fewC. globosum ascospores germinate in the absence of nutrients in the formof complex media was suggested as a possible explanation for its poorperformance.

With the exception of the lower fungi and yeasts, the most importantstructural component of the fungal cell wall is chitin (Lopez-Romero andRuiz-Herera, 1986). In the fungal cell wall, polymers of chitin areembedded in an amorphous matrix composed of β-1,3-glucans and aresusceptible to degradation by chitinases in the thin primary wall formedat the growing tip of the hyphae. Ordentlich et al. (1988) in in vitrostudies found that chitinolytic enzymes produced by the bacteriumSerratia marcescens caused degradation of 60% of the hyphal tip cells ofSclerotium rolfsii, a soil-borne pathogen.

Chitin has been used as a soil amendment for controlling plant parasiticnematodes and soil-borne pathogens with some success. The addition ofchitin to soil stimulates the growth of bacteria, actinomycetes, andfungi capable of producing chitinolytic enzymes (Brown et al., 1979;Godoy et al., 1983; Mitchell and Alexander, 1962). Godoy et al. foundthat the addition of chitin to soil stimulated the growth of organismscapable of degrading chitin, a component of the middle layer of eggshells in tylenchoid nematodes, through the production of chitinases.The study supported earlier results by Mian et al. (1982) thatdemonstrated the development of a particular soil microflora in responseto the addition of chitin. Many fungi, including species of Aspergillus,Chaetomium, Fusarium, and Verticillium, showed enhanced growth after theaddition of chitin to the soil and have been shown to be activelyinvolved in cyst and egg wall degradation of species of Heterodera andMeloidogynee (Godoy et al., 1982; Godoy et al., 1983; Morgan-Jones andRodriguez-Kabana, 1981; Ownley-Gintis et al., 1982).

In regard to the control of soil-borne fungal pathogens, Chet et al.(1986) have shown that chitin amendments stimulate the growth of thefungus Trichoderma harzianum which produces enzymes capable of degradingthe cell walls of Sclerotium rolfsii, Rhizoctonia solani, and Fusariumspp., giving it great potential as a means for controlling soil-borneplant pathogenic fungi.

Each of the approaches to microbiological biocontrol of plant diseasesdiscussed above has demonstrated only a limited success. In particular,it is noted that long term survival and reproduction of themicroorganisms was not shown to be enhanced by these approaches, despitethe number of years that biocontrol has been considered an ecologicallyand commercially important goal.

SUMMARY OF THE INVENTION

One aspect of this invention provides a method of controlling thepopulation of a first microorganism at a foliar locus by preferentiallyenhancing the population of a second microorganism at said locus,comprising applying to the locus an amount of a durable, substantiallywater-insoluble, polymeric substrate which is preferentially biodegradedby said second microorganism with respect to said first microorganism,whereby growth of said second microorganism is substantiallypreferential to that of said first microorganism. In a preferredembodiment, the polymeric substrate comprises a polysaccharide andfurther comprises a binder which increases the durability of thepolymeric substrate. In another preferred aspect, the method furthercomprises applying an inoculum of the second microorganism to saidlocus. Preferred polymers serve in this embodiment as food sources forthe desirable microflora.

Another aspect of this invention provides a method of controlling thepopulation of a first microorganism at a foliar locus by preferentiallyenhancing the population of a second microorganism at said locus,comprising applying to the locus an amount of a durable, substantiallywater-insoluble polymeric substrate effective as a selective habitatenhancer which substantially preferentially potentiates growth of saidsecond microorganism. In a preferred embodiment, the polymeric substrateis a polysaccharide, and further comprises a binder which increases thedurability of the polymeric substrate. In another preferred aspect, themethod further comprises applying an inoculum of the secondmicroorganism to said locus. Preferred polymers serve in this embodimentas physical protection for the desirable microflora.

Still another aspect of the invention provides a method of enhancing thepopulation of a microorganism at a foliar locus, comprising applying tothe locus an amount of a durable, substantially water-insolublepolymeric substrate which is biodegraded by the microorganism, wherebygrowth of the microorganism is potentiated. In a preferred aspect, thepolymeric substrate is applied in admixture with a binder whichincreases its durability.

A fourth aspect of this invention provides a method of enhancing thepopulation of a microorganism at a foliar locus, comprising applying tothe locus an amount of a durable, substantially water-insolublepolymeric substrate effective as a selective habitat enhancer whichpotentiates growth of said microorganism. In a preferred embodiment, thepolymeric substrate is a polysaccharide, and further comprises a binderwhich increases the durability of the polymeric substrate.

A fifth aspect of this invention provides a durable foliar habitatmodifier, comprising (a) a water-resistant, weather resistant polymericsubstrate, (b) a binder which increases the durability of the habitatmodifier, and (c) a microorganism whose growth is enhanced by thedurable foliar habitat modifier. In a preferred aspect, the modifierfurther comprises one or more of a surfactant, a buffering agent, adispersant, a humectant, a UV absorber, or an IR reflector.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanying drawingswherein:

FIG. 1 shows the effects on epiphytic actinomycetes of chitinFormulation with and without the addition of Bacillus cereus;

FIG. 2 shows the effect of buffer (CaCO₃ ⁻), Clandosan and chitin onpopulations of actinomycetes on peanut leaves;

FIG. 3 shows the effect of buffered and unbuffered chitin formulationson peanut early leafspot disease severity using a subject rating scalewhere 1.0=no disease and 2.0=20% of leaves infected by Cercosporaarachidicola;

FIG. 4 shows the effects of two bacteria (Bacillus cereus orCurtobacterium flaccumfaciens) applied alone (with Soy-dex, a dryingoil/sticker), with Soy-dex and buffers (formulation controls) or withSoy-dex, buffers, and Clandosan, on peanut leafspot disease severity;

FIG. 5 shows the numbers of Bacillus cereus cells isolated from fieldgrown peanut leaves 1 wk after application at 10⁸ cells/ml;

FIG. 6 shows the effects of formulation and applied bacteria onpopulations of epiphytic bacteria at early season (70 days) and lateseason (90 days). Treatments with the same letter are not significantlydifferent (P=0.05);

FIG. 7 shows a scanning electron micrograph of Curtobacteriumflaccumfaciens and other naturally occurring microorganisms colonizingchitin 5 days after application;

FIG. 8 shows a scanning electron micrograph of abnormal, pitted hyphacolonized by Bacillus cereus on peanut leaf treated with I-chitin(industrial chitin) formulation+Bacillus cereus 5 days afterapplication; 3,400X;

FIG. 9 shows a scanning electron micrograph of untreated peanut leafsurface with typical healthy fungal hypha; 2,000X;

FIG. 10 shows cellulose alone and in combined formulation withChaetomium globosum spores on the colonization by the fungus of appleleaf surfaces using scanning electron microscopy for visualization;

FIG. 11 shows biological control of sooty mold of apple fruits byChaetomium globosum as affected by the addition of nutrient polymers;and

FIG. 12 shows biological control of flyspeck of apple fruits byChaetomium globosum as affected by the addition of nutrient polymers.

DETAILED DESCRIPTION OF THE INVENTION

This invention thus provides a preferred method for biological controlof pathogens of above-ground plant parts whereby indigenous orexogenously applied organisms are provided with a durable selectivehabitat enhancer comprising a durable, substantially water-insolublepolymer that serves as a source of food and/or of physical protection.This invention similarly is applicable to methods for biological controlemploying microbial bioherbicides and bioinsecticides.

Previous attempts at biocontrol failed in part because of a harsh andnonsupportive foliar environment; this disadvantage is overcome by theapplication of this invention. In the prior art attempts, the preferredspecies failed to actually thrive and grow stably at the foliar locus.Invariably, the prior art applied microorganisms either alone or incombination with watersoluble amendments, primarily in the form of foodsupplements. These included water-soluble monomeric sugars, urea, whey,and components of microbiological media which are easily washed off theleaves by water in the form of rain or irrigation. However, the foliaramendments of this invention are durable, so that necessaryreapplications of the foliar amendments are infrequent, and are requiredprimarily to cover new growth, with the concomitant reduction indisturbance of the foliar ecosystem, once enhanced, as well as costreduction.

The mechanism by which the enhancement of this invention occurs can varywidely, including, but not limited to, physical effects such asenhancement of the sheltering aspects of the habitat which favor thepreferred species, and metabolic effects such as preferentialutilization of the habitat enhancer as a food source.

The mechanism of enhancement of preferential growth of the preferredspecies, preferably over that of the pathogenic species, may then haveits effects through several secondary effects, including, but notlimited to, selection pressure resulting from competition for limitedresources, production of selective antibiotics, lytic enzymes or toxinsby the antagonist, e.g., fungicides, insecticides, phytotoxins, etc.,direct parasitism of the pathogen by the preferred species, induction ofplant disease, stimulation of the host plant resistance response, etc.No matter what the mechanism is, however, the end result is that thepreferred species, by benign competitive antagonism and/or activelydetrimental antagonism of the species whose growth is to be controlled,is provided with an environmental selective advantage due to theenhancement of its habitat at the foliar locus. Often, of course, therewill be "tertiary" end-result effects flowing from these mechanisms,e.g., enhanced growth of a crop, control or killing of undesiredvegetation, etc. Also included in this invention will be instances wherethe enhanced growth of a desired microorganism and/or the slowed growthor death of an undesired microorganism is the end result. Furtherincluded are instances where the end result to be achieved is theenhanced growth of a microorganism, irrespective of any effects on anyother microorganism. These effects include production by the preferredspecies of plant growth hormones and bioinsecticides and detoxificationof plant pesticides. Thus, this invention provides a method for controlof disease, insects, vegetation, etc., without the application of toxic,persistent or otherwise environmentally undesirable chemicals.

The preferred species may be endogenous to the plant phylloplane, or maybe applied to the plant, concurrently or sequentially, with the durableselective habitat enhancer. In either case, the species whose growth isto be preferred may be selected by one of skill in the art from anyknown species, e.g., one which can antagonize the growth of the plantpathogen (including, for example, an insect or nematode pathogen) whichis to be controlled. If the preferred species is one which is alreadypresent at the foliar locus, it can be selected by one skilled in theart by knowledge of its presence, and what would provide a selectiveadvantage to it, which, if not known precisely, can easily be determinedby routine and conventional screening techniques in view of thisdisclosure. If the microorganism is to be added exogenously, it maysimilarly be selected by one of skill in the art by choosing from knownspecies, e.g., antagonists to a pathogen or insect whose growth is to becontrolled, or by routinely and conventionally screening known speciesin order to find a preferred antagonist. Examples of suitableantagonistic species known in the art for antagonizing particular knownplant foliar pathogens are shown in Table 1. Other examples of suitablepathogen-antagonist pairs can be found in Rogers, 1989. Previousattempts to use these antagonists to control the cited diseases have hadlimited success, however, due to the difficulty of maintaining stablepopulations of the antagonist in the phylloplane, as mentioned.

                  TABLE 1                                                         ______________________________________                                        Plant    Pathogen    Antagonist  Reference                                    ______________________________________                                        Wheat    H. sativum  Unk. bacterium                                                                            Simmonds, 1947                                                    isolated from                                                                 wheat                                                    Tree stumps                                                                            F. annosum  P. gigantea Artman, 1972                                 Tobacco  A. alternata                                                                              P. capecia  Spurr, 1981                                  Peanut   Cercospora  P. capecia  Spurr, 1981                                  Peanut   Cercospora  P. capecia and                                                                            Bailey and                                                        B. thuringiensis                                                                          Spurr, 1984                                  Geranium P. pelargonii-                                                                            B. subtilis Rytter and                                            zonalis                 Lukezic et                                                                    al., 1986                                    Cacao    M. roreri   Pseudomonas Jimenez et                                                                    al., 1986                                    Snap bean                                                                              B. cinerea  T. hamatum  Nelson and                                                                    Powelson, 1988                               Grape    B. cinerea  Trichoderma Gullino and                                           and P. viticola         Garibaldi,                                                                    1986                                         Corn     "Damping off"                                                                             C. globosum Kommedahl et                                                                  al., 1981                                    Apple    Apple scab  C. globosum Cullen et al.,                                                                1984                                         ______________________________________                                    

Other combinations of pathogen and antagonist will be apparent to one ofskill in the art, or can be routinely determined by one of such skill.Preferred will be particular strains which can be routinely isolatedfrom known sources or can be purchased from various commercialsuppliers.

Suitable microorganisms whose growth is to be enhanced include, but arenot limited to, bacteria, algae, yeasts, actinomycetes and fungi havingthe desired characteristics, e.g., having suitable antagonisticcharacteristics, including, for example, suitable metabolic pathways forthe polymeric substrate, opportune growth characteristics for theparticular plant and environment, and production of an appropriateantibiotic or lytic enzyme, elicitor, etc. for the particular pathogento be controlled. Suitable genera include Chaetomium, which are fungi;Bacillus, which are gram-positive bacteria; Enterobacterium, which aregram-negative bacteria; Curdobacterium, which are Coryneform bacteria;these exemplify a broad range of suitable genera. Other suitablemicroorganisms whose growth is to be enhanced include, but are notlimited to, engineered microorganisms, e.g., derived from the same typesdiscussed above, which are constructed so as to have several concomitantfavorable characteristics, as described above, e.g., engineeredselective ability to use the amendments of this invention (e.g., tometabolize a particular substrate) and/or engineered ability to producea secondary effect as discussed above.

Similarly, it is possible to preferentially enhance the growth ofmicroorganisms which produce, or which can be engineered to produce,toxins, for example, Bt (Bacillus thuringiensis toxins) in order to killparasitic plant insects, either by infecting the insects or by poisoninginsects which ingest the toxin either by eating the microorganismsthemselves or foliar surfaces contaminated with the toxin. Othercontemplated equivalents include preferential growth of microorganismswhich produce, or are engineered to produce, phytotoxins (biologicalherbicides) which are directed at selectively killing or inhibiting thegrowth of the detrimental plants on which they are growing, whileselectively not harming other plants, and of microorganisms whichproduce insecticides, nematicides, elicitors, hormones, e.g., plantgrowth hormones, etc. Still another possibility is the preferred growthof microorganisms which can detoxify conventional pesticides which maybe present on the foliar surface, or on the edible fruits, due to theuse of these pesticides in the conventional way. Thus, this aspect ofthe invention contemplates the preferred growth of pesticide detoxifyingmicroorganisms at various times and for various reasons; for example,just prior to harvesting, so as to decrease the toxic effects of theseagents upon the animals eating the plant products. These uses areexamples of embodiments wherein the effects on "other" microorganismsmay be irrelevant.

As used herein, the terms "foliar" and "phylloplane" relate to allabove-ground, aerial parts of the plant, especially the leaves, but alsoincluding the shoots, bark, trunk, stems, flowers, pods and fruits. Itexcludes surfaces contacting the soil; for example, the roots and otherunderground parts of the plant such as tubers.

Suitable plants to which the durable selective habitat enhanceraccording to this invention can be applied include any agriculturallyimportant plants, as well as decorative and home garden plants. Theseinclude, but are not limited to, monocots, dicots, gymnosperms,evergreens, ferns, mosses and lichens.

More generally, it is possible to use the method according to thisinvention to treat any above-ground surface to antagonize the growth ofundesirable microorganisms. For example, buildings and other surfacescontaining habitats consistent with growth, such as grout and metalroofs, metal sculptures, roof tiles, shingles, and swimming pool decks,may be treated with the durable selective habitat enhancer of thisinvention in order to prevent the growth of undesirable microorganismssuch as molds, algae, mosses, lichens and bacteria which causedeterioration or cosmetic damage to these surfaces, etc.

The term "durable" as used herein means long-lasting at the desiredsite, e.g., weather-resistant and waterresistant, to a sufficient degreethat the preferential growth of the selected-for microorganism is of adegree effective to cause the desired end-effect. This desired effectmay be, e.g., population disenhancement or suppression of the specieswhose growth is to be controlled, or population enhancement of a speciesto be benefitted, or some other secondary or end-result effect asdescribed herein. Population effects will generally be, e.g., about25-50%, preferably 100%, especially orders of magnitude more or lessthan the original level (depending on the desired effect). Thus, thedesired effect may also be, e.g., the amelioration of disease caused bythe microorganism whose growth is to be controlled to acceptable levels,preferably comparable with levels of amelioration achievable withconventional pesticides. Similarly, when the desired effect isinsecticidal or herbicidal, "durable" means environment-resistant, e.g.,weather-resistant and water-resistant, to a sufficient degree that thepreferential growth of the selected-for microorganism is of a degreeeffective to cause the desired insecticidal or herbicidal effect. Ingeneral, as applied to the selective habitat enhancer at the foliarlocus, the term "durable" means that the material substantially remainson said locus for, e.g., >1 week, and preferably >2-3 weeks. Even morepreferably, the material will not substantially be removed by thenatural environment, irrigation, or other erosive events during thistime period, or preferably during the entire relevant period of time,e.g., the growing season or the pathogen-sensitive time period for theparticular plant in a given year.

The term "selective habitat enhancer" as used herein means an agentwhich, when applied to a foliar locus, modifies the microfloralenvironment at said locus such that the ecology of the microfloralenvironment is substantially changed in a selective manner with respectto one microorganism or more, but not equally for all microorganisms,e.g., by physical means or by metabolic amendment which ispreferentially biodegraded and utilized by the preferred species.

By "physical means" is meant that the selective habitat enhancer doesnot function primarily by biodegradation of a provided agent but ratherfunctions primarily, e.g., by selectively providing physical protection(shelter) or a suitable microclimate or microenvironment favorable toone or more of the desired species in the phylloplane such that thegrowth thereof is preferentially enhanced, e.g., over that of a specieswhose growth is to be controlled. The "shelter" can be from predation,from adverse effects of UV and/or IR radiation, from weather (e.g.,wind, precipitation, aridity (by providing a local relatively highhumidity)), etc. Other agents can also be included to augment thiseffect, such as agents which modify UV and IR effects, for example, IRreflectors, e.g., light colored or white materials, and UV absorbers,e.g., molecules containing carbon-carbon double bonds; agents whichmodify humidity effects, for example, humectants; buffers; etc. Thusthese physical effects generally operate via the foliar coating providedby this invention which provides or enhances protection againstweathering by means such as a fold, raised surface, indentation,crevice, etc., in the coating, or which filters out UV and/or IRwavelengths from radiation impinging on microorganisms entrapped withinsuch loci, or which impedes evaporation of water or attracts theaccumulation of water. Thus, generally, the term "habitat enhancer", asit refers to the physical effects provided, means a material ormaterials which selectively enhances the growth of a preferred speciesby providing a suitable physical environment favoring its growth andreplication.

By "metabolic amendment" is meant that the selective habitat enhancerfunctions primarily by biodegradation of a provided agent as a selectivefood source for the preferred microorganism. Thus, for example, thepolymeric substrate which is applied may be one which is subject todegradation primarily by means of enzymes which the preferredmicroorganism possesses, and thus represents a source of nutrients notavailable, or available only to a lesser extent, to the microorganismwhose growth is to be controlled, and thereby the growth of thepreferred microorganisms is enhanced over that of the microorganismwhose growth is to be controlled. Thus, generally, the term "habitatenhancer", as it refers to the metabolic effects provided, means amaterial which is selectively degraded as a food source by and therebyenhances the growth of a preferred species by providing a suitableselective nutritional source favoring its growth and replication.

In either case, the enhancement of growth of the preferred species mayresult in one or more effects which substantially affect the microfloralcomposition of the foliar locus, especially with respect to the specieswhose growth is to be controlled. Non-limiting examples of these effectsinclude the possibility that the durable selective habitat enhancer mayfunction by enhancing the growth of the preferred microorganism, whichin turn causes selective pressures well known to those of skill in theart; these selective pressures can result in the preferred speciesadvantageously competing with other species for other limited nutrientresources, to the detriment of the other species. These limited nutrientsources at the foliar locus include, e.g., leaf exudates and leachatesand deposited organic matter such as pollen and aphid honeydew. Anotherpossibility is that the preferred species whose growth is enhancedproduces an antibiotic that either inhibits the growth or replication ofother organisms in the phylloplane, or that is lethal to them. Forexample, many bacteria and fungi produce antibiotics that are effectiveagainst plant pathogens. Still another possibility is direct parasitismof the pathogen by the preferred species, which is enabled to gain afoothold in the foliar environment by having a selective advantage assupplied by the habitat enhancer, either as food or shelter.

The preferred species may consist of a single species or multiplespecies They may co-exist in order to enhance each other's growth, orthey may co-exist independently to antagonize one or more microbialplant pathogens. They may be endogenous to the foliar locus or appliedsimultaneously with the durable selective habitat enhancer, orsequentially, either prior to or after the application of the durableselective habitat enhancer.

The term "polymeric substrate" as used herein means materials composedof straight-chain and/or branchedchain polymers. These include, but arenot limited to, polysaccharides, polypeptides, especially proteins, andpolynucleotides. These may be natural biomolecules or syntheticpolymers. They also include polymers which may or may not bemetabolizable by a given microorganism. Thus, if the polymeric substrateis to function as a durable selective habitat enhancer primarily byphysical means, as described above, it is preferred that the polymer benon-metabolizable. However, if the polymer functions primarily as a foodamendment for the preferred species of microorganism, it is preferredthat the polymer be metabolizable by the preferred species,preferentially to its metabolism by the species whose growth is to becontrolled.

Excluded from the scope of polymeric substrates of this invention arecommercial slow-release polymeric formulations wherein a desiredingredient is absorbed or adsorbed or covalently bonded to a polymer. Insuch prior art embodiments, the polymer per se is not biodegraded butmerely serves as a source of the desired ingredient.

"Polymers" in this invention generally refer to materials having adegree of polymerization (number of polymerized monomer units) of atleast 20, preferably 100-250 monomeric units, typically of a weightaverage molecular weight of about 4000-10,000 daltons. Moreparticularly, in the case of polymers whose monomeric units aresubstantially soluble in water, polymers according to this inventioninclude materials which are polymerized to the extent that they aresubstantially insoluble in water. The substrates may of course containinnocuous byproducts such as low molecular weight materials from thesame monomers.

By "substantially insoluble" is meant a solubility in water at 20° C. ofat most 10%, preferably <1%, most preferably essentially completelyinsoluble in water. Thus, the materials of this invention, when in theform to be applied to the plants, will be substantially in the form of asuspension or a sol, rather than a solution.

Suitable polysaccharide substrates include, but are not limited to,polysaccharides which can be biodegraded by some microorganisms.Non-limiting examples of the wide range of suitable polysaccharidesinclude chitin, cellulose, β-1,3-glucans, carrageenan, poly(galacturonicacid) such as pectin, lignin and its derivatives, polylevulan,hemicellulose, xylan and mucopolysaccharides, and derivatives thereof,e.g., sulfated, nitrated, aminated, and phosphorylated derivatives.Nitrated and aminated derivatives are preferred.

It is preferred that the polymeric substrate of this invention,especially when it is a polysaccharide, be applied in a form that isconducive both to application protocols and/or to make the polymersenzymically available to the microorganisms as a habitat enhancer. Thisform may be achieved through the controlled hydrolysis of a complex,especially crystalline, polymer by an acid, a base or an enzyme to aless compact conformation. Thus, for example, in the case of chitin,hydrolysis of the highly crosslinked crystalline form of chitin to along-chain branched form which is amorphous and fluffy in appearance ispreferred. The hydrolysis should be of a limited extent such that themajority of the polymeric substrate remains substantially insoluble inwater.

Chitin is a particularly preferred polymeric substrate. It is comprisedprimarily of poly(1→4)-N-acetyl-D-glucosamine, and is a polysaccharidefound in nature in the cell walls of fungi, nematode egg shells, andinsect and crustacean exoskeletons. It is preferred that the chitin usedin this invention be hydrolyzed prior to application, to the extentnecessary to break tertiary crystalline structure and thereby formamorphous material, e.g., by the use of an acid, base, or enzymatically,using conventional protocols, e.g., as described in the examples. Thusit is preferred that the chitin be hydrolyzed to the extent of, forexample, having a weight average molecular weight of about 4000-10,000daltons, as described above.

Cellulose, poly-β-(1→4)-glucose, is also a naturally occurringpolysaccharide. Only a few organisms, including representatives ofbacteria, actinomycetes and fungi contain cellulolytic enzymes.

Other suitable polymeric substrates include polypeptides andpolynucleotides, as well as terpenes, long chain fats and oils and otherlike polymers, and combinations thereof.

Suitable physical sheltering polymers useful in this invention includeany of the above-listed polymers which cannot be biodegraded by therelevant microorganism.

The term "binder" as used herein means a material which enhances thedurability of the polymeric substrate upon the foliar locus. A suitablebinder will thus adhere the agent to the desired surface and helpprevent . it from washing or weathering off the leaves. Suitable bindersinclude, but are not limited to, drying oils and latex derivatives.Suitable drying oils include, for example, soybean oil, tung oil andlinseed oil, as well as any conventional binder known in the art ofherbicides and insecticides which are not toxic to the preferredmicroorganisms. Suitable binders of these types can be routinelydetermined by one of ordinary skill in the art. Other agents which maybe useful as binders include polypectates and gums. Especially in thecase of latexes being used as binders, there may be present othermaterials in the form of copolymers with the latex such asorganosilicones, such as, for example, SILWET® (Union Carbide, Danbury,Conn.). It is further noted that, especially in the case when oilsand/or fats are used as the binder, and especially when these oils orfats contain 16-24 carbons per molecule, the binder may simultaneouslyserve as both a binder and a substrate for the growth of themicroorganism.

In addition to drying oils, the formulations of this invention mayinclude other adjuvants which potentiate the adhesion of the durableselective habitat enhancer to the foliar locus or achieve otherdesirable effects such as surfactants, buffering agents, dispersants,wetting agents, etc. These materials are well known in the art, andtheir suitability for a particular application of this invention can beroutinely determined, as above.

Other optional adjuvants which enhance the growth of the preferredorganism are also contemplated. These can include, but are not limitedto, humectants such as, for example, carboxymethyl cellulose, forincreasing the moisture content of the polymeric substrate;IR-reflecting materials, for example, materials which impart a white,IR-reflective surface to the habitat enhancer, to prevent damage fromsolar radiation; UV-absorbing materials such as, for example UVnul® orlignin, also to prevent damage from solar radiation; surfactants,particularly organosilicone types, or others which are of low toxicityto microorganisms; and other adjuvants such as buffers, as mentionedabove. These adjuvants can be added in amounts of 0.005 to 5.0% of theformulation, more or less also being useful.

In particular, the addition of UV-absorbing agents is well-known as amethod of stabilizing UV-sensitive materials such as polymers,especially polymeric and oligomeric UV absorbers and especially thosecontaining double-bonded carbon atoms. A low weight percentage of suchagents (0.01 to 2.0% of the formulation, more or less also being useful)distributed uniformly throughout the material to be protected canprovide dramatic and long-lasting reductions of levels of UV radiationwithin and/or underneath the material. Polymeric agents capable ofperforming in this fashion are reviewed extensively by D. Bailey and O.Vogl, J. Macromol. Sci. Rev. Macromol. Chem. C14(2), 267-293 (1976) andD.A. Tirrell, Polymer News 7:104-110 (1981). Examples of suchUV-absorbing agents suitable for this invention include but are notlimited to microbially non-toxic amounts of:

2-hydroxy-4-methacryloxybenzophenone/ethylene homopolymer;

2-hydroxy-4-methacryloxybenzophenone/vinylidene chloride homopolymer;

2-hydroxy-4-methacryloxybenzophenone/alkyl acrylate homopolymer;

2-hydroxy-4-acryloxybenzophenone/ethylene homopolymer;

2-hydroxy-4-acryloxybenzophenone/vinylidene chloride homopolymer;

2-hydroxy-4-acryloxybenzophenone/alkyl acrylate homopolymer;

2-hydroxy-4-methacryloxybenzophenone/ethylene copolymer;

2-hydroxy-4-methacryloxybenzophenone/vinylidene chloride copolymer;

2-hydroxy-4-methacryloxybenzophenone/alkyl acrylate copolymer;

2-hydroxy-4-acryloxybenzophenone/ethylene copolymer;

2-hydroxy-4-acryloxybenzophenone/vinylidene chloride copolymer;

2-hydroxy-4-acryloxybenzophenone/alkyl acrylate copolymer;

2-hydroxy-4-acryloxybenzophenone/styrene copolymer;

2-hydroxy-4-methacryloxyacetophenone/ethylene homopolymer;

2-hydroxy-4-methacryloxyacetophenone/vinylidene chloride homopolymer;

2-hydroxy-4-methacryloxyacetophenone/alkyl acrylate homopolymer;

2-hydroxy-4-acryloxyacetophenone/ethylene homopolymer;

2-hydroxy-4-acryloxyacetophenone/vinylidene chloride homopolymer;

2-hydroxy-4-acryloxyacetophenone/alkyl acrylate homopolymer;

2-hydroxy-4-methacryloxyacetophenone/ethylene copolymer;

2-hydroxy-4-methacryloxyacetophenone/vinylidene chloride copolymer;

2-hydroxy-4-methacryloxyacetophenone/alkyl acrylate copolymer;

2-hydroxy-4-acryloxyacetophenone/ethylene copolymer;

2-hydroxy-4-acryloxyacetophenone/vinylidene chloride copolymer;

2-hydroxy-4-acryloxyacetophenone/alkyl acrylate copolymer;

2-hydroxy-4-acryloxyaceto-phenone/styrene copolymer.

The durable habitat enhancers of this invention can also be mixed withfungicides, bactericides, acaricides, nematicides, insecticides, orother biologically active compounds in order to achieve desired resultswith a minimum expenditure of time, effort and material. Amounts ofthese biologically active materials added for each part by weight of thecomposition of this invention may vary from 0.05 to 25 parts by weight.Suitable agents of this type are well known to those skilled in the art.Some are listed below:

Fungicides:

methyl 2-benzimidazolecarbamate (carbendazim)

tetramethylthiuram disulfide (thiuram)

n-dodecylguanidine acetate (dodine)

manganese ethylenebisdithiocarbamate (maneb)

1,4-dichloro-2,5-dimethoxybenzene (chloroneb)

methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl)

2-cyano-N-ethylcarbamoyl-2-methoxyiminoacetamide (cymoxanil)

N-(trichloromethylthio)tetrahydrophthalimide (captan)

N-(trichloromethylthio)phthalimide (folpet)

dimethyl 4,4'-(O-phenylene)bis(3-thioallophanate)(thiophanate-methyl)

2-(thiazol-4-yl)benzimidazole (thiabendazole)

aluminum tris (O-ethyl phosphonate)(phosethyl aluminum)

tetrachloroisophthalonitrile (chlorothalonil)

2,6-dichloro-4-nitroaniline (dichloran)

N-(2,6-dimethylphenyl)-N-(methoxyacetyl)alanine methyl ester (metalaxyl)

cis-N-[1,1,2,2-tetrachloroethyl)thio]cyclohex-4-ene-1,2-dicarbioximide(captafol)

3-(3,5-dichlorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide (iprodione)

3-(3,5-dichlorophenyl)-5-ethenyl-5-methyl-2,4-oxazolidinedione(vinclozolin)

kasugamycin

)-ethyl-S,S-diphenylphosphorodithioate(edifen-phos)

4-(3-(4-(1,1-dimethylethyl)phenyl)-2-methyl)-propyl-2,6-dimethylmorpholine(Fenpropimorph)

4-(3,4(1,1-dimethylethylphenyl)-2-methyl)propylpiperidine (Fenpropidine)

1-[[(bis(4-fluorophenyl)methylsilyl]methyl]-1H-1,2,4-triazole(flusilazole)

2-p-chlorophenyl-2-(1H-1,2,4-triazol-1-ylmethyl)-hexanenitrile(myolobutanil)

(±)-1-[2-(2,4-diohlorophenyl)-4-propyl-1,3-dioxolan-2ylmethyl]-1H-1,2,4-triazole(propiconazole)

N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboxamide(prochloraz)

(RS)-2,4'-difluoro-α-(1H-1,2,4-triazol-1-ylmethyl)-benzhydryl alcohol(flutriafol)

1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1yl)butanone(triadimefon)

1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol(triadimenol)

(2RS,3RS)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol(diclobutrazol)

Bactericides:

tribasic copper sulfate

streptomycin sulfate

oxytetracycline

Acaricides:

senecioic acid, ester with 2-sec-butyl-4,6-dinitrophenol (binapacryl)

6-methyl-1,3-dithiolo[2,3,B]quinonolin-2-one (oxythioquinox)

2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol(dicofol)

bis(pentachloro-2,4-cyclopentadien-1-yl) (dienochlor)

tricyclohexyltin hydroxide (cyhexatin)

hexakis(2-methyl-2-phenylpropyl)distannoxane (fenbutin oxide)

Nematocides:

2-[diethoxyphosphinylimino]1,3-diethietane (fosthietan)

S-methyl1-(dimethylcarbamoyl)-N-(methylcarbamoyloxy)-thioformimidate(oxamyl)

S-methyl 1-carbamoyl-N-(methylcarbamoyloxy)thioformimidate

N-isopropylphosphoramidic acid, O-ethylO'-[4-(methylthio)-m-tolyl]diester (fenamiphos).

Insecticides:

3-hydroxy-N-methylcrotonamide(dimethylphosphate)ester (monocrotophos)

methylcarbamic acid, ester with 2,3-dihydro-2,2-dimethyl-7-benzofuanol(carbofuran)

O-[2,4,5-trichloro-α-(chloromethyl)benzyl]phosphoric acid,O',O'-dimethyl ester (tetrachlorvinphos)

2-mercaptosuccinic acid, diethyl ester, S-ester with thionophosphoricacid, dimethyl ester (malathion)

phosphorothioic acid, O,O-dimethyl, O-p-nitrophenyl ester (methylparathion)

methylcarbamic acid, ester with α-naphthol (carbaryl)

methyl N-[[(methylamino)carbonyl]oxy]ethanimidothioate (methomyl)

N'-(4-chloro-o-tolyl)-N,N-dimethylformamidine (chlordimeform)

O,O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidyl)phosphorothioate(diazinon)

octachlorocamphene (toxaphene)

O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN)

cyano(3-phenoxyphenyl)-methyl-4-chloro-α-(1-methylethyl)benzeneacetate(fenvalerate)

(3-phenoxyphenyl)methyl(±)-cis,trans-3-(2,2-dichloroethenyl)-2,2,-dimethylcyclopropanecarboxylate(permethrin)

dimethylN,N'-[thiobis(N-methylimino)carbonyloxy]]bis[ethanimidothioate](thiodicarb

phosphorothiolothionic acid, O-ethyl-O-[4-(methylthio)phenyl]-S-n-propylester (sulprofos)

α-cyano-3-phenoxybenzyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (cypermethrin)

cyano(3-phenoxyphenyl)methyl4-(difluoromethoxy)-α-(methylethyl)benzeneacetate (flucythrinate)

O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphorothioate (chlorpyrifos)

O,O-dimethyl-S-[[(4-oxo-1,2,3-benzotriazin-3-(4H)-yl)methyl]phosphorodithioate(azinphos-methyl)

5,6-dimethyl-2-dimethylamino-4-pyrimidinyl dimethyl carbamate(pirimicarb)

S-(N-formyl-N-methylcarbamoylmethyl)-O,O-dimethylphosphorodithioate(formothion)

S-2-(ethylthioethyl)-O,O-dimethyl phosphiorothioate (demeton-S-methyl)

α-cyano-3-phenoxybenzylcis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate(deltamethrin)

cyano(3-phenoxyphenyl)methyl ester ofN-(2-chloro-4trifluoromethylphenyl)alanine (fluvalinate).

The durable selective habitat enhancers of this invention can be appliedby fully conventional techniques known for applying formulations, e.g.,liquids to foliage.

Possible formulation types include dusts, granules, pellets,suspensions, emulsions, wettable powders, emulsifiable concentrates andthe like. Many of these may be applied directly. Sprayable formulationscan be extended in suitable media and used at spray volumes of from afew liters to several hundred liters per hectare. High strengthcompositions are primarily used as intermediates for furtherformulation. The formulations, broadly, contain about 0.1% to 99% byweight of active ingredient(s) and optionally (a) about 0.1% to 2%surfactant(s) and (b) about 1% to 99.9% solid or liquid inertdiluent(s). More specifically, they will contain these ingredients inthe following approximate proportions:

    ______________________________________                                               Active     Weight Percent*                                                    Ingredient Diluent(s)                                                                              Surfactant(s)                                     ______________________________________                                        Wettable 20-90        0-74      1-10                                          powders and                                                                   water                                                                         dispersible                                                                   granules                                                                      Oil       3-50        40-95     0-15                                          suspensions,                                                                  emulsions,                                                                    (including                                                                    emulsifiable                                                                  concentrates)                                                                 Aqueous  10-50        40-84     1-20                                          suspension                                                                    concentrates                                                                  Dusts     1-25        70-99     0-5                                           Granules and                                                                           0.1-95         5-99.9  0-15                                          pellets                                                                       High strength                                                                          90-99         0-10     0-2                                           compositions                                                                  ______________________________________                                         *Active ingredient plus optionally one of a surfactant or a diluent equal     100 weight percent.                                                      

Lower or higher levels of active ingredient can, of course, be presentdepending on the intended use and the physical properties of theingredients. Higher ratios of surfactant to active ingredient aresometimes desirable, and are achieved by incorporation into theformulation or by tank mixing.

Typical solid diluents are described in Watkins, et al., "Handbook ofInsecticide Dust Diluents and Carriers", 2nd Ed., Dorland Books,Caldwell, N.J., but other solids, either mined or manufactured, may beused. The more absorptive diluents are preferred for wettable powdersand the denser ones for dusts. Typical liquid diluents and solvents aredescribed in Marsden, "Solvents Guide," 2nd Ed., Interscience, New York,1950. Solubility under 0.1% is preferred for suspension concentrates;solution concentrates are preferably stable against phase separation at0° C. "McCutcheon's Detergents and Emulsifiers Annual", MC PublishingCorp., Ridgewood, N.J., as well as Sisely and Wood, "Encyclopedia ofSurface Active Agents", Chemical Publishing Co., Inc., New York, 1964,list surfactants and recommended uses. All formulations can containminor amounts of additives to reduce foaming, caking, corrosion,microbiological growth, etc.

The methods of making such compositions are well known. Solutions, e.g.,intermediate compositions, are prepared by simply mixing theingredients. Fine solid compositions are made, e.g., by blending and,usually, grinding as in a hammer or fluid energy mill. Water dispersiblegranules may be produced, e.g., by agglomerating a fine powdercomposition (see, for example, B. Cross and H. Scher, "PesticideFormulations", ACS Symposium Series 371, American Chemical Society,Washington, D.C., 1988, pp. 251-159). Suspensions are prepared, e.g., bywet milling (see, for example, Littler, U.S. Pat. No. 3,060,084).Granules and pellets may be made, e.g., by spraying the active materialupon preformed granular carriers or by agglomeration techniques. SeeJ.E. Browning, "Agglomeration", Chemical Engineering, Dec. 4, 1967, pp.147ff. and "Perry's Chemical Engineer's Handbook", 5th Ed., McGraw-Hill,New York, 1973, pp. 8-57ff.

For further information regarding the art of formulation, see forexample:

H.M. Loux, U.S. Pat. No. 3,235,361, Feb. 15, 1966, Col. 6, line 16through Col. 7, line 19 and Examples 10 through 41;

R.W. Luckenbaugh, U.S. Pat. No. 3,309,192 March 14, 1967, Col. 5, line43 through Col. 7, line 62 and Examples 8, 12, 15, 39, 41, 52, 53, 58,132, 138-140, 162-164, 166, 167 and 169-182;

H. Gysin and E. Knusli, U.S. Pat. No. 2,891,855, Jun. 23, 1959, Col. 3,line 66 through Col. 5, line 17 and Examples 1-4;

G.C. Klingman, "Weed Control as a Science", John Wiley and Sons, Inc.,New York, 1961, pp. 81-96; and

J.D. Pryer and S.A. Evans, "Weed Control Handbook", 5th Ed., BlackwellScientific Publications, Oxford, 1968, pp. 101-103.

Suitable amounts of the durable selective habitat enhancer can beroutinely determined, and application of these amounts can be achieved,for example, by spraying the plants until the point of runoff of thesolution. Other methods of application will also be suitable, dependingupon the technique chosen.

The timing for applications can be routinely determined and adapted fora particular plant, pathogen and antagonist by one of ordinary skill inthe art. Thus it may be preferable for a particular plant to be treatedprophylactically when there is a known threat of a particular pathogen,or it may be preferable to wait until there are early signs ofinfection. It may be preferable to apply the durable selective habitatenhancer at a particular point in a particular plant's life cycle; forexample, at leaf budding, abscission or setting of fruit. In othercases, application may be timed by the weather or time of year, whichfavors the growth of certain plant pathogens. Still further,applications may be single or multiple, depending upon the durability ofthe durable selective habitat enhancer and, if it is a biodegradablefood source, for example, for the preferred species, the rate at whichit is being used up. Other factors in determining the frequency ofreapplication include the growth rate of new foliage on the plants, therate of environmental erosion, the frequency of infection events, thefrequency of insect flights, etc. These factors are routine, andapplication intervals can be determined analogously to standard methodsused in the agricultural industry.

The durable selective habitat enhancer of this invention is preferablyapplied, e.g., in colloidal suspensions in concentrations of, forexample 0.1 to 5.0%, w/w of water. These suspensions can generallycontain, for example, from 0.2 to 1.0% of the habitat enhancer (forexample, preferably about 0.5 for chitin and 1.0% for cellulose), 0.001to 0.02% of the binder, and 0.001 to 0.1% of the other adjuvants.

If desired, the durable selective habitat enhancer can be produced andstored in concentrate form of 2 to 25%, w/w, suspended in water, and canbe diluted with water to the appropriate concentration for applicationjust prior to use. Alternatively, the enhancer can be prepared inwettable powder form or dispersable granule form and made up with waterat the time of application.

It will be appreciated that the concentration of the inoculum ofmicroorganism which can optionally be applied exogenously to the plantsbefore, during or after treatment with the durable selective habitatenhancer will vary depending upon the type of microorganism, the fieldconditions, how it is applied, etc. Suitable concentrations can beroutinely determined by one of skill in the art. Suitable culturingconditions for growing the large quantities needed of any particularmicroorganism are similarly highly conventional, including suitablenutrient media, liquid and solid substrate fermentation equipment,temperatures, methods of determining cell concentrations, etc. Themicroorganisms may be applied as a metabolically active form or in sporeform. The microorganisms preferably will be added to the durableselective habitat enhancer just prior to application to the plants, ifthey are coapplied. Otherwise, they can be applied to the plants, e.g.,as a spray at the appropriate concentrations suspended in, e.g., abinder, either before or after application of the habitat enhancer ofthis invention. If the microorganisms are applied prior to theapplication of the habitat enhancer, however, it is preferred that theybe applied only shortly before the habitat enhancer is applied, in orderfor the full benefit of the protective and/or nutrient selectivity ofthe habitat enhancer to be obtained. It is also possible that themicroorganism can be present in the suspensions, concentrates or powderscontaining the habitat enhancers of this invention which can be stored,if the suspensions or concentrates are frozen, or at room temperature ifthe microorganism is present in an inactive form, such as in the form ofspores contained in the powders.

A preferred formulation of, for example, chitin and a drying oil into acolloidal suspension for foliar application as a durable selectivehabitat enhancer according to this invention resulted in durablepersistent residues capable of modifying the population of extantepiphytic microorganisms and also enhancing the survivability of appliedchitinolytic bacteria. The ability to manipulate populations ofepiphytic microorganisms by the application of, for example, chitinindicated a potential for biocontrol. A model pathogen/host system,Cercospora arachidicola on peanut, was studied, with and without theapplication of an exogenous microorganism. The exogenously appliedmicroorganisms chosen for this system was a Bacillus cereus strainoriginally isolated from field-grown peanuts treated with the colloidalchitin formulation and selected as a potential antagonist because of itschitinolytic activity, as well as its ability to colonize field-grownpeanut leaves. Curtobacterium flaccumfaciens was isolated fromgreenhouse-grown peanuts and selected as a potential antagonist becauseit was a prolific chitinase producer.

The use of an inexpensive, industrial grade chitin (Clandosan®) resultedin more dramatic shifts in populations of epiphytic microflora thanpurified chitin (P-chitin). The incorporation of a calcium carbonatebuffer into the formulation further accentuated shifts in microbialpopulations and enhanced the survival of the two exogenously appliedbacterial antagonists. A 60% reduction in disease severity compared tountreated controls occurred on plants treated with buffered Clandosan®+B. cereus, which exhibited better survivability and disease suppressionoverall than C. flaccumfaciens. B. cereus was reisolated frominoculated, Clandosan®-treated plots at populations of 3×10⁴ cell/gramof leaf tissue 7 days after application. Viewing the leaf surface withan electron microscope, Bacillus cells could be seen dividing andgrowing.

Another preferred formulation comprising cellulose +Soydex® (85% soybeanoil:15% surfactant; Helena Chemical Co., Memphis, TN) similarlyexhibited good durability and weatherability on foliar surfaces.Applying amorphous cellulose to the leaves created physical environmentswhich improved survival of microbial antagonists, e.g., by protectingthem from UV and IR irradiation and desiccation. These polymers can alsoform a physical barrier which prevents infection by foliar pathogens.Another approach involved the application of the cellulolytic fungalantagonist Chaetomium globosum as a means of biological control ofpathogens, which was compared with and without the addition of a durableselective habitat enhancer according to this invention. The severity ofapple diseases flyspeck (Zygophiala jamaicensis) and sooty blotch(Gloeodes pomigena) were significantly reduced by the treatmentsaccording to the invention. Cellulose +C. globosum reduced the number offlyspeck infection loci by 63% while cellulose alone reduced the numberby 36% over the untreated control. Chaetomium survival and growth wereincreased significantly by the addition of cellulose (which probablyfunctioned as a source of food), but not by the addition of chitin;indigenous phylloplane populations were suppressed by C. globosumgrowth.

Disease suppression can result from the lysis of fungal cell walls bythe chitinase-producing antagonists and by enriched populations ofnative chitinolytic microorganisms. Scanning electron microscopy showedthat the applied bacteria colonized and digested chitin particles andfungal hyphae. The nature of the chitin formulation on the leaf surfaceprovides potential physical environments suitable for colonization bymicroorganisms, and forms a physical barrier that may prevent thegermination of spores of pathogenic microorganisms landing on it. Directantibiosis through the production of inhibitory compounds by the appliedbacteria can also occur.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and unless otherwise indicated, allparts and percentages are by weight.

The entire disclosure of all applications, patents and publications,cited above and below, are hereby incorporated by reference.

EXAMPLES Example 1: Addition of Metabolic Amendment

a. Effect on resident foliar microorganisms

b. Effect on applied microorganism

Methods

A colloidal suspension of chitin was prepared by acid hydrolysis of 3 kgof flaked crustacean chitin (Sigma Chemical Co.) in 23 L of concentratedHCl. Rodriguez-Kabana et al. (1983). The mixture was allowed to standfor 2 hours at 28° C. until dissolved, then diluted with 114 L of water,stirred, and allowed to settle overnight. The supernatant was siphonedoff and discarded. Fresh water was added, and the suspension was mixedthoroughly and allowed to resettle. The rinsing process was repeatedtwice daily for 7-10 days until the pH exceeded 2.0. The concentratedchitin suspension was then filtered through a 0.7 mm mesh screen, placedin 1 gal containers, and stored at 4° C. The chitin concentration of thefinal suspension was determined by drying 1 ml samples in a tared watchglass at 60° C. for 10 h. Dry weight was determined to the fifth decimalplace.

Preliminary Test and Bacterial Culture

A 1% solution of colloidal chitin was applied until just before runoffto 5-wk-old, field-grown peanuts, in a non-replicated test to evaluateseveral spray adjuvants, e.g., stickers and spreaders, for the desiredcoverage and weathering characteristics. Visual monitoring of thedeposits and an evaluation regarding the ease with which the depositscould be rubbed off were performed. After 2 weeks, samples of thephylloplane microflora were collected using leaf wash and dilution platetechniques, and cultures were grown on nutrient agar containing 0.2%chitin. A chitinolytic, spore-forming Bacillus cereus Cohn was selectedfrom these cultures and transferred to nutrient broth containing 0.4%chitin. Broth cultures were placed on a rotary shaker for 5 days, afterwhich they were harvested by filtration through a Whatman No. 2 filter.Cells were rinsed from the filter with sterile water, and the turbidityof the solution was optically adjusted to 1.0-5.0×10⁸ cells/ml.

Field Trials

Concentrations of chitin, B. cereus, and a fungicide control (commercialstandard) were tested for effects on epiphytic microbial populationsunder field conditions at the Auburn University Agronomy Farm, Auburn,Alabama. "Florunner" peanuts were planted in rows 0.8 m apart. Theaqueous concentrations of chitin tested were 0.0%, 0.1%, 0.2%, 0.5%, and1.0%. Control plots were treated with chlorothalonil (500 gl/L flowable,Bravo 500L®, Fermenta Plant Protection, Painesville, Ohio) at 2.3 L/ha.A soybean oil-surfactant blend (SOSB) (85%:15% soybean oil:surfactant,Soydex® Helena Chemical Co., Memphis, Tennessee) was added as anadjuvant to all treatments at 0.25%. The chitin and fungicide treatmentswere applied with or without B. cereus beginning 56 days after planting.The 12 treatments were arranged as single row plots 1.5 m long in arandomized complete block design, each with three replications. Alltreatments were applied until just before runoff (approximately 650L/ha). One hundred ml of the concentrated bacterial cell suspension waadded to 900 ml of water and applied to field plots subsequent to theapplication of the organic amendment. Applications were made withhand-held spray pump sprayers on a seven-day schedule beginning Jul. 20,1986, and continuing for nine weeks (Sep. 25).

Sampling and Isolation of Organisms

Epiphytic populations were estimated by randomly collecting 10 g of leaftissue from upper, middle, and lower portions of the peanut canopybeginning two weeks after the first treatment and continuing atfour-week intervals throughout the season. The leaf samples were addedto 100 ml of sterile tap water in 250 ml Erlenmeyer flasks. One drop ofsurfactant (Tween 20) was added as a wetting agent, and the flasks wereplaced on a rotary shaker at 100 rpm for 1 hour. The leaf wash solutionwas diluted by adding 1 ml of wash solution to a tube containing 9 ml ofsterilized tap water. A second ten-fold dilution was made, and 0.1 ml ofeach dilution was transferred aseptically to 90 mm Petri platescontaining 20 ml of the following medium: nutrient agar +0.2% chitin(CNA) to estimate actinomycete and bacterial populations, as well astheir chitinolytic activity. Three replicate plates of CNA per plot wereinoculated with leaf washates at dilutions of 10⁻³ g leaf tissue/mlwater and 10⁻⁴ g leaf tissue/ml water. All plates were incubated at 30°C. for 5 days. Plate counts were expressed as colony-forming units/g wetleaf tissue (CFU/g). Colonies were counted using a dark field colonycounter, and each was assumed to have developed from a single propagule.Fungi and dominant bacteria were identified to genus, and mean colonycounts of the three replicate plates for each medium were determined.

Statistical Analysis

Statistical analyses were performed using microcomputer SAS generallinear models procedures (SAS User's Guide, 1985). Treatment means wereevaluated for least significant differences utilizing a Student T test.Unless otherwise stated, significant differences were at the 0.05 levelof probability.

Results Preliminary Test and Culture

The preliminary test indicated that the colloidal chitin suspensioncontaining the soybean oil-surfactant blend (SOSB) exhibited excellenttenacity, remaining on the leaf surface for several weeks whilewithstanding heavy rain, wind, and intense solar radiation. Bacilluscereus was selected for use in subsequent tests from numerouschitinolytic bacteria isolated from the preliminary test plants andlater was identified by fatty acid analysis (Microbial ID, Inc., Newark,Del.). This bacterium exhibited rapid growth in culture, as well asmoderate chitinase production, and inhibition of adjacent colonies ofsome bacteria and fungi.

Field Test

Populations of epiphytic actinomycetes, bacteria, fungi, and yeasts wereaffected differently by application of chitin and/or the presence orabsence of the applied chitinolytic bacteria B. cereus. Both qualitativeand quantitative differences in organisms isolated on CNA fromchitin-treated and untreated leaves were found. Midseason samplescollected in early September at 100 days after planting showed adramatic and highly significant increase in actinomycete populationswith the addition of chitin (FIG. 1). Populations did not varysignificantly in 0.2-1.0% chitin treatments in the absence of B. cereus.However, the addition of B. cereus resulted in a significant increase inactinomycete populations, with the highest numbers occurring with theaddition of 0.2% chitin. The addition of B. cereus to the formulationcontrol had no effect on actinomycete levels.

The late season isolation at 130 days after planting indicated a generaldecline in actinomycete numbers on plants treated with higherconcentrations of chitin, as compared to those on plants treated withthe formulation control and 0.1% chitin. The addition of B. cereusincreased actinomycete numbers significantly in the presence of 1.0%chitin but decreased them significantly when added to the formulationcontrol.

Neither the application of chitin nor the chitinolytic B. cereusbacterium caused any significant changes in populations of epiphyticbacteria at midseason. However, there was a general trend (P<0.10) forB. cereus to cause a decline in the numbers of epiphytic bacteria. Latein the season, the rate of chitin caused a highly significant (P<0.01)increase in total epiphytic bacteria populations. In the presence ofchitin, the addition of B. cereus caused a further increase inpopulations of epiphytic bacteria (disregarding the applied B. cereus).

The incidence of early leafspot, caused by C. arachidicola, wasdetermined by an objective rating system (data not presented). Diseaseseverity on fungicide-treated peanuts was lower than those treated withthe chitin formulation or controls. Disease severity was notsignificantly different among chitin treatments, indicating that thisportion of the leaf microflora was not affected by the formulationstested.

Discussion

The addition of SOSB to the colloidal chitin suspension provided it withextended weatherability such that the spray interval of 7-10 days was afunction of the need to treat new foliage rather than the loss ofresidue, which remained visible for periods of greater than one month.The extended period of time that the chitin suspension remained on theleaf facilitated the development of a greatly increased microflora(0.5-1.0 log₁₀) in general, and also of a large population ofchitinolytic microflora, indicating the bio-availability of thecolloidal chitin. Bacillus cereus was selected from this modifiedmicroflora as a suitable organism to apply to leaf surfaces insubsequent tests for several reasons: (1) B. cereus is a Gram-positive,spore-forming bacterium, providing it with environmental insensitivity:(2) cultures on chitin agar showed it to be a rapid-growing, chitinaseproducer that occurred in moderate numbers on peanut leaves, and (3)Bacillus spp. are known to produce a variety of antibiotics which wasillustrated by the inhibition of some bacteria and numerous fungi by B.cereus in culture, suggesting that this may also occur on the leafsurface (data not presented).

For bacterial antagonists applied to foliage, the major cause of celldeath is desiccation (Leban et al., 1965). Moisture is also one of themajor limiting factors in the growth and reproduction of the naturallyoccurring epiphytes. The unusually hot and dry conditions during thesummer of 1986 probably caused the total populations of epiphyticorganisms to be lower than during a cooler, wetter year. However, thedata indicated overall increases in the populations of epiphyticactinomycetes, bacteria, and fungi with the addition of chitin.Microbial isolations indicated that the added B. cereus was surviving atpopulations of up to 1 ×10⁴ CFU's/g wet leaf tissue seven days afterbeing applied to field plots.

Bacteria and actinomycetes have been reported to be the predominantcolonizers of leaf tissue early in the growing season, while fungi andyeasts become dominant later in the season (Blakeman, 1985). The resultspresented here support these general patterns of succession. They weresignificantly influenced, however, by the addition of B. cereus andcolloidal chitin, alone or in combination. Actinomycetes, which areconsidered to be the predominant microorganisms involved in chitindegradation and chitinase production in soil (Veldkamp, 1955) and whichare stimulated by the addition of chitin to soil (Goday et al., 1983),also were stimulated by the addition of chitin and B. cereus to the leafat midseason (FIG. 1). This increase was probably due to ability todegrade chitin through the production of chitinolytic enzymes. Thetendency for populations of epiphytic bacteria to decline in thepresence of B. cereus and chitin at midseason could be due to severalfactors. These include the bacterium having a competitive advantage forchitin as a food source and/or the production of antibiotics or otherinhibitory compounds.

Overall results of population studies indicated that epiphyticmicroorganisms are affected differentially by the addition of largenumbers of one component of the population in combination with aselective food source.

Example 2: Control of Foliar Pathogens by Addition of MetabolicAmendments Introduction

Cercospora arachidicola, the causal fungus of early leafspot of peanut,is a destructive pathogen capable of causing up to 50% pod yield losswhen not controlled with fungicide sprays (Smith, 1984). Managementpractices can aid in the reduction of initial inoculum, but primarycontrol of early leafspot is through application of chemical fungicidessuch as chlorothalonil throughout the growing season.

Methods Greenhouse Test and Bacterial Culture

A test was conducted in the greenhouse to select a formulation buffer ofan appropriate pH for bacterial growth and enzymatic activity, but thatwas not phytotoxic. Four buffers (potassium phosphate pH 5.4, sodiumpropionate pH 6.4, calcium hydroxide pH 11.5, and calcium carbonate pH8.0) were tested on greenhouse grown peanuts (data not presented). Twochitinolytic bacteria, Bacillus cereus Cohn isolated from field grownpeanuts in 1986, and Curtobacterium flaccumfaciens (Hedges) Collins &Jones, isolated from greenhouse grown peanuts treated with bufferedcolloidal chitin and SOSB, were identified using fatty acid analysis(Microbial ID, Inc., Newark, Del.), and cultured in 2.8 liter Fernbachflasks containing 1 L of nutrient broth +0.4% chitin. Cultures wereplaced on a rotary shaker for 5 days and harvested by centrifugation at5000 g for 10 minutes.

Field Trials

Two field tests were conducted at the Auburn University Agronomy Farm,Auburn, Ala. "Florunner" peanuts were planted May 5, 1987, using 0.8 mrow spacing. In test a, two chitin preparations were compared. The firstpreparation utilized purified chitin (P-chitin) and was made usingflaked crustacean chitin (Sigma Chemical Co.). P-chitin was used in the1986 field trial and prepared as described in Example 1. The secondchitin preparation, industrial chitin (Clandosan), was prepared from aground chitin-protein complex (McCandliss et al., 1985), as describedabove. A 3×2 factorial design consisting of three levels of chitin whichwere: (1) a 1% suspension of P-chitin, (2) a 1% suspension of Clandosan,and (3) no chitin (control). Each level of chitin was applied in twoforms: (1) buffered with a 1.0% suspension of CaCO₃ at pH 8.0 and (2)unbuffered. SOSB was added at 0.25% as a spray adjuvant to alltreatments with the exception of the unbuffered, no chitin control. Thesix treatments were arranged using single row plots (2.5 m) inrandomized complete blocks with six replications.

In test b, two chitinolytic bacteria were used in conjunction withClandosan which was diluted to 1.0% w/v chitin and buffered using asuspension of CaCO₃ (1 gram/100 ml water) to pH 8.0. A soybeanoil-surfactant blend (88:15, Soydex, Helena Chemical Co., Memphis,Tenn.) was added at 0.25% as a spray adjuvant. The chitin preparationwas compared to untreated plots as well as to a formulation controlconsisting of CaCO₃₊ 0.25% SOSB using a 3×3 factorial design. The threeformulations included (1) Clandosan, (2) formulation control, and (3)untreated control. The three levels of applied organism were (1) noadded organism, (2) B. cereus, and (3) C. flaccumfaciens. An aqueoussuspension of approximately 10⁸ bacterial cells/ml +0.25% soybeanoil-surfactant blend (SOSB) was applied to field plots. The ninetreatments were arranged using single-row plots (3 m long) in randomizedcomplete blocks with six replication per treatment.

Treatments were applied to peanuts in both tests using hand-heldcompressed air sprayers until just before leaf runoff (approximately 650L/ha). A seven-day spray schedule was followed beginning June 19 andcontinuing through Aug. 29, 1987.

Disease Evaluations

Evaluations of early leafspot were made on both field tests concurrentlyusing two methods. A subjective rating in which 1.0=no disease and2.0=20% or more of total leaf area diseased was performed at cropmaturity (85 days after planting). An objective rating (total lesions)was performed in which all early leafspot lesions occurring on leafletsat nodes 4, 7, and 10 from the shoot apex were counted.

Statistical Analysis

Statistical analyses were performed using microcomputer SAS (SAS User'sGuide, 1985), general linear models and orthogonal comparisonprocedures. Unless otherwise stated, differences were significant at the0.05 level of probability.

Results Test a: Formulation Experiment

Greenhouse experiments to examine the performance of several buffersindicated that a suspension of calcium carbonate at pH 8.0 was notphytotoxic to peanut foliages, and was within an acceptable pH range forbacterial growth and enzyme production and activity (Rodriguez-Kabana etal., 1983). Excellent coverage and tenacity were exhibited by theformulation when calcium carbonate and SOSB were added to the colloidalsuspensions of P-chitin and Clandosan. Scanning electron micrographs ofuntreated peanut leaves, and leaves treated with buffered colloidalClandosan formulation illustrated an altered leaf surface topographywith the addition of the chitin formulation.

Microbial isolations at 70 days (early) and 95 days (late) afterplanting indicated that numbers of epiphytic actinomycetes (FIG. 2) werehighest on Clandosan treatments at both dates, with significantly higherlevels in the buffered treatments early in the season. Orthogonalcomparisons indicated increased populations of actinomycetes (P=0.01) onClandosan treated plants compared to either P-chitin or controltreatments. Buffered P-chitin was superior to the unbufferedformulation, indicating that buffering has some impact on reducingdisease severity. This is supported by orthogonal analysis of unbufferedchitin treatments compared to buffered chitin treatments which indicateda reduction in disease severity (P<0.04) with buffering using thesubjective disease rating system (FIG. 3).

Test b: Addition of Antagonists

Differences between treatments were apparent when the total lesionnumbers of three leaves were used (FIG. 4). A 60% reduction in diseaseseverity was recorded between untreated control plants and plantstreated with Clandosan and B. cereus. Orthogonal comparison of allClandosan-containing treatments to all formulation control treatmentsshowed a highly significant (P<0.01) decrease in disease severity withthe addition of Clandosan. Using orthogonal analysis, a highlysignificant (P<0.01) decrease in late season defoliation was evidentwhen Clandosan treatments were compared to formulation treatments, anduninoculated Clandosan was compared to Clandosan +B. cereus or Clandosan+C. claccumfaciens.

Discussion

The objective rating system (number of lesions/3 leaves) was used toquantify differences between treatments. This system indicated thatuntreated control plants had four times as many lesions as plantstreated with buffered Clandosan. Further, the buffered control treatmentsuppressed disease as well as the unbuffered chitin treatmentsindicating that the buffer and adjuvant mixture contributed to theeffects of chitin in suppressing disease. This effect could be due to amore suitable pH for the growth of antagonists such as actinomycetes andfungi, which increased on buffered Clandosan. Reduced disease severityin the formulation control treatment could also be due to the SOSBacting as a metabolic amendment or possibly the toxic effect of thesurfactants on the pathogen (Backman et al., unpublished result).

In Test b, the subject rating system indicated that the addition of B.cereus to untreated plants provided the same level of diseasesuppression as the chitin treatments, suggesting that B. cereus actedantagonistically to the pathogen. Both B. cereus and C. flaccumfaciens,as well as the formulation control, suppressed disease in the absence ofchitin when percent infection was calculated. (The total lesion ratingwas more effective in elucidating differences between treatments.) TheClandosan formulation +B. cereus was the most effective treatment insuppressing disease, with a 60% reduction in the number of lesionscompared to the untreated control. Using this evaluation system, ageneral trend for the addition of organisms to lessen lesion numbersbecame apparent. The numbers of added organisms were low when comparedto total epiphytic bacteria, indicating that disease suppression onplots treated with bacteria was accomplished by a subdominant componentof the microflora, i.e., approximately 2.4% of the total microflora 1week after application. In the case of B. cereus, this response wassignificantly enhanced with the addition of chitin as were populationlevels of B. cereus at seven days following treatment, indicating thatthe organism was utilizing the amendment in some way.

Example 3: Electron Micrographic Studies of the Effects of Added Chitinand Added Microorganisms at the Foliar Locus Methods

Leaves sampled from the experiment described in Example 1 were collectedrandomly, as were leaves from Example 2.

Electron Microscopy

Peanut leaf samples from all treatments were fixed for scanning electronmicroscopy (SEM) in formalin-acetic acid-alcohol fixative (FAA 5%, 5%,70%) for 24 hours in a vacuum desiccator. Specimens were then dehydratedin a graded series of ethanol (50%-100%), critical point dried, mountedon aluminum stubs, sputter-coated with gold/palladium, and examinedusing an ISI Model SS40 scanning electron microscope at 5 kv.

Isolation of Organisms

Epiphytic populations were estimated by systematically collecting 10 gof leaf at 70 and 95 days after planting and were dilution plated toselective media as described in Example 1. The medium used was nutrientagar +0.2% chitin (CNA) to estimate populations of bacteria, includingadded bacteria, as well as chitinolytic activity. Three replicate platesper plot of CNA inoculated with dilutions of 10⁻³ g leaf tissue/ml waterand 10⁻⁴ g leaf tissue/ml water. All plates were incubated at 30° C. for5 days. Plate counts were expressed as colony-forming units/g wet leaftissue (CFU/g).

Results Leaf Ecology

Microbial isolation, as well as observation by scanning electronmicroscopy, indicated that both species of applied bacteria (B. cereusand C. flaccumfaciens) were surviving the application process and werereproducing on chitin-treated leaves at least seven days afterapplication. FIG. 5 indicates that background populations of B. cereuson untreated plots were approximately 100 CFU's/gram leaf tissue. Oneweek after application to chitin-treated leaves, B. cereus was isolatedat 3×10⁴ CFU's/gram leaf tissue. Curtobacterium flaccumfaciens wasrecovered from inoculated I-chitin (I-chitin=industrialchitin=Clandosan) treated plots at populations of 1×10² CFU's/gram leaftissue five days after application (data not presented). Numbers ofnaturally occurring epiphytic bacteria (excluding added bacteria) oneach of the nine treatments at early and late season isolations arerepresented in FIG. 6. At both sampling dates, chitin greatly increased(two- to three-fold) numbers of total epiphytic bacteria, while therewas a general trend for the addition of either B. cereus or C.flaccumfaciens to reduce numbers of naturally occurring epiphyticbacteria, particularly at the early date. Orthogonal analysis indicatedthat there was a significant increase (P<0.01) in actinomycetepopulations in Clandosan treatments compared to formulation controltreatments.

Scanning electron micrographs of peanut leaves treated with the I-chitinformulation inoculated with C. flaccumfaciens showed typical Coryneformcells coionizing chitin in large numbers (FIG. 7). Further, theyillustrated an altered surface topography where chitin had been applied.Colonization of I-chitin by applied C. flaccumfaciens and indigenousmicroorganisms also was apparent (FIG. 7), which indicated both growth(dividing cells) and metabolism of the substrate (fossil-likeimpressions on the chitin where cells had previously existed).Micrographs of chitin-treated, B. cereus inoculated leaves at five daysafter application revealed abnormally pitted fungal hyphae colonized bybacterial cells, some of which appeared to be dividing (FIG. 8), incontrast to untreated control leaves which were colonized by moretypical fungal hyphae (FIG. 9).

These data indicate that the addition of applied biological controlorganisms along with a selectively available metabolic amendment(chitin) results in improved survival of the organisms and increasedantagonism to pathogens. This benefit appears to arise through chitinutilization as a metabolic amendment and by improved environment forindigenous and applied organisms.

Example 4: Effect of Cellulose and Chitin on a Fungal Antagonist of anApple Pathogen Introduction

Andrews et al. (1983) screened 50 microorganisms isolated from the applephyllosphere for antagonism to Venturia inaequalis. These were thenranked according to efficacy based on three in vitro and three in vivotests: growth on nutrient agar, germination, and germ tube lengths onagarose-coated slides; and lesion size, overall symptom development, andconidial production on infected leaves. The best and most consistentlyantagonistic organism was C. globosum.

The severity of apple scab was consistently reduced by up to 90% ingrowth chamber studies when C. globosum ascospores are applied to appleseedlings along with conidia of the pathogen, V. inaequalis (Andrews etal., 1983). However, frequent field applications over three seasonsreduced disease by only 0-25% (Boudreau and Andrews, 1987). The methodof antagonism of c. globosum to V. inaequalis and other pests issuspected to be antibiosis. Although Chaetomium globosum has been usedeffectively in small field trials as a biological control agent againstseedling blights, seed rots, and the bean seed fly, it has failed toexhibit effective and sustained apple scab suppression outside ofcontrolled environments. This failure is typical of past foliar diseasebiological control work in general. This example utilizes the same hostand antagonist to determine if results can be improved with the polymerfood base technology.

Methods Acid Hydrolysis of Cellulose

Powdered cellulose (Celufil, non-nutritive bulk, United StatesBiochemical Corporation) was mixed with phosphoric acid (85%, Fisherbrand, ACS grade) in a blender using a high-speed setting at aconcentration of approximately 25 g of cellulose per 1.0 liters of acid(unpublished method of R. Rodriguez-Kabana). The resultant suspensionwas allowed to stand at room temperature for 30 minutes withintermittent stirring. Then the acid-cellulose mixture was placed in a115 liter plastic garbage can with roughly 95 1 of tap water. Thecontents of the garbage can were then stirred, and thehydrolyzed-cellulose precipitate was allowed to settle. When thesupernatant was clear, it was siphoned off, and the garbage can wasrefilled with water and stirred vigorously. This process of siphoningand refilling was repeated several times to "wash" the hydrolyzedcellulose and raise the pH toward neutral.

When the pH reached about 6.0, the supernatant was drained, and thecellulose sediment that remained was blended at high speed for severalminutes to disintegrate any clumps. The suspension was strained using a25 mesh screen, resuspended in excess distilled water, and allowed tosettle. When the supernatant became clear, it was decanted, and theremaining liquid suspension was conserved as the final product.

Fungal Culture and Spore Production

Chaetomium globosum strain NRRL 6296 was selected for use in thisexperiment because of its demonstrated suppressive activity through theproduction of antifungal compounds to the apple scab pathogen, Venturiainaequalis (Boudreau and Andrews, 1987), and its rapid and abundantsporulation. Permanent cultures of C. globosum were maintained on lacticacid acidified potato dextrose agar (pH=4.5) and cellulose agar.

Cellulose agar was prepared by mixing 500 ml of 0.5% hydrolyzedcellulose suspension with 500 ml of tap water. To this mixture, 1.0 gpotassium nitrate, 0.5 g potassium phosphate (dibasic), 0.2 g magnesiumsulfate, and 30 g agar were added. The resultant solution was autoclavedand poured 0.5 cm deep into 8"×12"×4"alcohol-sterilized plastic cakepans, inoculated with C. globosum ascospores, and sealed withalcohol-sterilized plastic lids. These culture flats were incubated at23° C. in sterile plastic garbage bags.

Preliminary Tests

Preliminary tests were conducted on the ability of C. globosum to growon cellulose or chitin as a sole carbon source. This was done byinoculating petri dishes of 0.25% cellulose agar, 0.25% chitin agar, orwater agar with C. globosum ascospores. These dishes were monitored over14 days for sporulation and hyphal growth. The cellulose and chitin agarplates were also monitored for cleared zones indicating hydrolysis ofthe carbon source around the C. globosum colonies.

Field Tests

Field tests were designed to evaluate the possibility of biologicallycontrolling apple scab (Venturai inaequalis), flyspeck (Zygophialajamaicensis), and sooty blotch (Gleodes pomigena) with Chaetomiumglobosum.

The biological disease control properties of Chaetomium globosum wereevaluated on trellised Early Red One apples on M26 rootstock at theNorth Alabama Horticultural Station, Cullman, Alabama. Thirty-six treeswere arranged into four replicates, which each contained ninetreatments: (1) chitin+C. globosum+N-acetylglucosamine (NAG)+Soydex, (2)chitin+NAG+Soydex, (3) cellulose+cellobiose+C. globosum+Soydex, (4)cellulose+cellobiose+Soydex, (5) C. globosum+Soydex, (6) Soydex, (7)untreated, (8) benomyl 50W (Benlate, du Pont, 1.19 kg/kl)+captan 50W(Chevron, 4.78 Kg/Kl)+Soydex, (9) C. globosum. All concentrations ofcellulose, chitin, Soydex were 0.5% (w/v, w/v, and v/v, respectively).Fertilization and insect control of apple trees were administeredfollowing the current industry standards.

All treatments were applied with 4 liter, hand-pumped tank sprayers toone-half of each tree; the treated half of each tree constituted oneplot, and the untreated halves acted as buffers between treatments.Treatments receiving an organic amendment and C. globosum spores weresprayed twice on each spray date: Once with C. globosum plus Soydex andonce with the organic amendment plus Soydex. This increased control overthe concentration of C. globosum spores applied.

Treatments began when the apple leaves emerged in the spring (April 4)and were reapplied every seven days. This schedule was maintained forfive weeks (until May 5), after which treatments were applied threetimes on a 14-day schedule until the trees reached terminal bud (June16). Thereafter, applications were made every 21 days until the testtermination on Aug. 3.

Plots were treated on the following dates: Apr. 4, Apr. 14, Apr. 21,Apr. 28, May 5, May 19, Jun. 2, Jun. 6, and Jul. 7. Mean daily maximumtemperatures, mean daily average temperatures, and mean daily averagerainfall data were collected throughout the season.

Samples of 25 apple fruits were collected from each of the 36 plots onAug. 8, 1988, and rated for sooty mold (Gloeodes pomigena) and flyspeck(Zygophiala jamaicensis) infection. Sooty mold was subjectively rated ona scale from 0.0 to 3.0: 0.0 represented no visible infection, 1.0represented a light infection on some fruits, 2.0 represented a heavyinfection on some fruits with most fruits infected to some extent, and3.0 represented a heavy infection on all fruits. Flyspeck levels weredetermined by counting the number of flyspeck infection loci or patcheson each apple fruit.

Inoculum Survival and Growth

Leaf samples were randomly collected from 12 of the 36 plots on Apr. 28immediately after treatments were applied for the fourth time. The 12sampled plots were four replicates from the following treatments:cellulose +cellobiose+Soydex, C. globosum+Soydex, and cellulose+cellobiose+C. globosum+Soydex. A small cork borer was used to randomlycollect three 0.5 cm leaf disks from three different leaves from eachplot sample. This ensured adequate sample representation and uniformsample size which facilitated more accurate comparisons amongtreatments. These leaf disks were then prepared for scanning electronmicroscopy which provided visual evidence of, and allowed for asubjective rating of, C. globosum survival and colonization of the leafsurface; any colonization would be from the first three treatmentapplications. The subjective rating scale was between 0.0 and 4.0: 0.0represented no C. globosum growth, 1.9 represented very few short hyphalstrands visible on the leaf surface, 2.0 represented a few long hyphalstrands and a moderate complement of short strands visible on the leafsurface, 3.0 represented many long hyphal strands and many short hyphalstrands visible on the leaf surface, and 4.0 represented a hyphal matcovering the leaf surface.

Results Preliminary Tests

Greenhouse experimentation demonstrated that 0.5% cellulose (w/v) with0.5% Soydex (v/v) provided the best leaf coverage and amendment tenacityto apples. It was also determined that the most complete coverage wasachieved by spraying plants to near runoff.

Evaluations of culture media demonstrated that Chaetomium globosum wascapable of limited hyphal growth but with abundant sporulation whengiven either 0.25% hydrolyzed cellulose or 0.25% hydrolyzed chitin as asole carbon source in a 2.0% agar medium. The fungus produced virtuallyno hyphae and did not sporulate when transferred to water agar plates.Chaetomium colonies produced cleared zones in both the chitin andcellulose agar.

Inoculum Survival and Growth

Scanning electron microscopy of the leaf disks clearly showed that C.globosum was extensively colonizing the cellulose deposits on the leafsurfaces seven days after the fourth treatment application. Subjectiveevaluations of leaf disks revealed that plots receiving only C. globosumascospores+Soydex had very limited Chaetomium growth or colonization ofthe leaf surface. Plots receiving only cellulose (FIG. 10) had a highercolonization rating than plots receiving only C. globosum ascospores,but this increase was not statistically significant. This may indicatethat there was a natural background of C. globosum since uninoculatedplots had C. globosum growth, or it may indicate that a low level ofinoculum spray drift occurred. The plots receiving both cellulose and C.globosum spores had a higher colonization rating than either of theother two treatments (alpha=0.05). No other cellulolytic or chitinolyticorganisms were detected.

Field Tests

When apples collected from each plot were subjectively rated for sootymold disease levels (FIG. 11), untreated samples had the highest ratingand were significantly different from all other treatments.Fungicide-treated plots had the lowest level of disease. However, soydexdid suppress sooty mold, reducing disease by more than 50 percent.Therefore, the level of disease suppression provided by each treatmentis partially due to Soydex, except in the case of C. globosum ascosporesalone, which contained no Soydex.

The number of flyspeck infection loci per fruit (FIG. 12) shows thatcellulose along significantly reduced disease (alpha=0.10) by 36% overthe untreated control, cellulose+C. globosum reduced disease by 63%, andthe fungicide reduced disease by the greatest margin, 93%.

Discussion

The addition of cellulose with Chaetomium globosum ascospores greatlyenhanced Chaetomium growth. This increase could be due to the creationof physical environments that provided microclimates favorable for C.globosum growth and/or the utilization of cellulose as a food base.

Cellulose and chitin both adhered well to the apple foliage and fruit,and both amendments were suppressive to fruit disease when appliedeither with without C. globosum ascospores. When cellulose was appliedwith C. globosum, the principal mode of disease suppression was probablyantibiosis. This is supported by the observation (from microscopy andleaf-wash dilution-plating) that C. globosum nearly eliminated theindigenous microflora.

The observation that C. globosum alone was not suppressive to eitherflyspeck or sooty blotch is similar to the results reported by Boudreauand Andrews (1987) in which C. globosum alone failed to reduce applescab in field tests.

Subjective ratings of sooty blotch on field grown apples confirmed thissuppression and suggested additive control and possibly synergismbetween the amendments and C. globosum. This control was especiallyapparent in the flyspeck infection loci counts where cellulose+C.globosum exhibited excellent flyspeck suppression.

The control exhibited by cellulose+C. globosum indicated that theconcept of selectively enhancing the growth of a suppressive organismthrough the creation of physical environments and through the provisionof nutrients was practical.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

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What is claimed is:
 1. A method of controlling the population of a firstmicroorganism at a foliar locus by preferentially enhancing thepopulation of a second microorganism at said locus, comprising applyingto the locus an amount of a durable, substantially water-insolublepolymeric substrate having a solubility in water (20° C. of at most 1%which has been selected for being preferentially biodegraded by saidsecond microorganism, whereby growth of said second microorganism issubstantially preferential to that of said first microorganism, andwherein said polymeric substrate is applied in admixture with a binderwhich increases its durability.
 2. A method of claim 1, furthercomprising applying an inoculum of the second microorganism to saidlocus.
 3. A method of claim 1, wherein the durability of the polymericsubstrate at the foliar locus is at least 2 weeks.
 4. A method of claim1, wherein the polymeric substrate is a polysaccharide, a protein or amixture thereof.
 5. A method of claim 4, wherein the polymeric substrateis cellulose, chitin, carrageenan, a β-1,3-glucan, or poly(galacturonicacid), or a mixture thereof.
 6. A method of claim 5, wherein thepolymeric substrate is chitin or cellulose.
 7. A method of claim 1,wherein the second microorganism is Bacillus subtilis, Bacillus cereus,Curtobacterium flaccumfaciens or Chaetomium globosum.
 8. A method ofclaim 1, wherein the second microorganism is endogenous.
 9. A method ofclaim 1, wherein the binder is a drying oil.
 10. A method of controllingthe population of a first microorganism at a foliar locus bypreferentially enhancing the population of a second microorganism atsaid locus, comprising applying to the locus an amount of a durable,substantially water-soluble polymeric substrate having a solubility inwater (20° C.) of at most 1% effective as a selective habitat enhancerwhich has been selected for substantially preferentially potentiatinggrowth of said second microorganism with respect to said firstmicroorganism, and wherein said polymeric substrate is applied inadmixture with a binder which increases its durability.
 11. A method ofclaim 10, wherein the polymeric substrate is applied in admixture with abinder which increases its durability.
 12. A method of claim 10, whereinthe habitat modifier functions by provision of a selective physicalenvironment for said second microorganism.
 13. A method of claim 10,further comprising applying an inoculum of the second microorganism tosaid locus.
 14. A method of claim 10, wherein the polymeric substrate iscellulose.
 15. A method of claim 13, wherein the second microorganism isBacillus subtilis, Bacillus cereus, Curtobacterium flaccumfaciens orChaetomium globosum.
 16. A method of enhancing the population of amicroorganism at a foliar locus, comprising applying to the locus andamount of a substantially water-insoluble polymeric substrate having asolubility in water (20° C.) of at most 1% which has been selected forbeing effective as a durable, selective habitat enhancer whichpotentiates growth of said microorganism, and wherein said polymericsubstrate is applied in admixture with a binder which increases itsdurability.
 17. A method of claim 16, wherein the habitat modifierfunctions by provision of a protective physical environment for saidmicroorganism.
 18. A method of claim 16, wherein the polymeric substrateis biodegraded by the microorganism.
 19. A method of claim 6, whereinthe polymeric substrate is chitin which has been hydrolyzed to a weightaverage molecular weight of from 4000 to 10,000 daltons.
 20. A method ofclaim 9, wherein the polymeric substrate is chitin.